KR20230064363A - Diglucoside of epigallocatechin gallate(EGCG) and method of producing the same - Google Patents

Abstract

The present invention relates to EGCG diglucoside represented by Chemical Formula 1 in which isomaltose is modified at the 4'-OH position of Epigallocatechin gallate (EGCG) and a method for preparing the same, and more particularly, It relates to a method for producing EGCG diglucoside by reacting EGCG and an isomaltose donor with an enzyme in an aqueous solution. EGCG diglucoside prepared by the method of the present invention shows a result of more than two times improved stability in an aqueous solution compared to EGCG, and can be used in various fields such as cosmetics, food, and pharmaceuticals.

Description

Epigallocatechin gallate diglucoside and method for producing the same {Diglucoside of epigallocatechin gallate (EGCG) and method of producing the same}

The present specification relates to epigallocatechin gallate diglucoside and a method for preparing the same.

Epigallocatechin gallate (hereinafter referred to as EGCG) is one of the four main catechins in green tea and has antibacterial properties. EGCG is known to have the strongest antioxidant activity among green tea catechins. In addition, it has been proven to show anticancer effects through interactions with various biochemical processes, and its utilization is very high.

However, EGCG is not soluble in water and has low stability in an aqueous solution, limiting its application to food, cosmetics, and pharmaceuticals. Therefore, a method of increasing the stability of EGCG has been studied, and a known representative method is a method of blocking interaction with other substances by low pH, low temperature, and chemical treatment. environment must be created. In addition, when the EGCG molecule is chemically modified, sugars are modified at various -OH ends of EGCG, so it is very difficult to obtain a specific molecule at a high rate.

KR10-2006-0063703

The present specification discloses a method for preparing EGCG diglucoside from EGCG at a conversion rate of 90% or more using a specific enzyme that does not require unnecessary chemical structural modification and additional chemical composition, and materials produced by the method.

In one aspect, the present invention relates to an EGCG glycoside compound in which 4'-OH of epigallocatechin gallate (EGCG) is substituted with isomaltose, a salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof to provide.

In one aspect, the present invention provides a method for preparing an EGCG glycoside compound, a salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof, comprising reacting EGCG and an isomaltose donor in the presence of an enzyme, wherein the enzyme is a glycosyl hydrolase enzyme, and provides a manufacturing method.

In one aspect, the method for producing EGCG diglucoside provided by the present invention can secure EGCG diglucoside with improved stability in an aqueous solution.

1 is a schematic diagram showing a process for preparing EGCG diglucoside.
Figure 2 shows the results of separating and purifying the enzyme reactant (a) and confirming the purity (b).
3 is a mass spectrometry chromatogram of EGCG diglucoside.
4 is a high-resolution mass spectrometry chromatogram of EGCG diglucoside.
Figure 5 shows the results of nuclear magnetic resonance analysis (2D HMBC) of EGCG diglucoside.
Figure 6 shows the results of accelerated persistence evaluation of EGCG diglucoside in aqueous solution.
Figure 7 is a graph comparing the lifespan of EGCG diglucoside in an aqueous solution (25 ° C).

Hereinafter, the present invention will be described in detail.

"Stereoisomers" as used herein means in particular optical isomers (eg, essentially pure enantiomers, essentially pure diastereomers or mixtures thereof), as well as conformational isomers. Conformation isomers (i.e. isomers that differ only in the angle of one or more chemical bonds), position isomers (particularly tautomers) or geometric isomers (e.g. cis-trans isomers) include

As used herein, "essentially pure", e.g., when used in reference to enantiomers or diastereoisomers, contains at least about 90%, preferably at least about 95%, of the specific compound exemplified by the enantiomer or diastereomer. , more preferably at least about 97% or at least about 98%, even more preferably at least about 99%, even more preferably at least about 99.5% (w/w).

In the present specification, the salt may be a pharmaceutically acceptable salt. As used herein, “pharmaceutically acceptable” means approval by the government or a regulatory body equivalent thereto that can be used in animals, more specifically in humans, by avoiding significant toxic effects when used in normal medical dosages. Received or approved, or listed in a pharmacopeia or otherwise recognized in a general pharmacopeia.

As used herein, "pharmaceutically acceptable salt" means a salt according to one aspect of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound.

As used herein, “salts thereof” or “pharmaceutically acceptable salts” are (1) formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) Benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-Toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2,2,2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert - acid addition salts formed with organic acids such as butylacetic acid, laurylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; or (2) a salt formed when an acidic proton present in the parent compound is substituted.

In the present specification, "hydrate" means a compound in which water is bound, and is a broad concept including an inclusion compound having no chemical bonding force between water and the compound.

As used herein, "solvate" refers to a higher order compound formed between solute molecules or ions and solvent molecules or ions.

In one aspect, the present invention relates to an EGCG glycoside compound in which 4'-OH of epigallocatechin gallate (EGCG) is substituted with isomaltose, a salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof to provide.

In an exemplary embodiment, the glycoside may be EGCG diglucoside represented by Formula 1 below.

[Formula 1]

In one aspect, the present invention provides an aqueous solution composition comprising an EGCG glycoside compound, a salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof.

In one exemplary embodiment, the composition may be a food, cosmetic, or pharmaceutical composition.

In one aspect, the present invention provides a method for preparing an EGCG glycoside compound, a salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof, comprising reacting EGCG and an isomaltose donor in the presence of an enzyme, wherein the enzyme is a glycosyl hydrolase-based enzyme, and provides a manufacturing method (FIG. 1).

The glycosyl hydrolase enzyme is a transglycosyltransferase, and performs a glucose transfer role.

In an exemplary embodiment, the glycosyl hydrolase enzyme is a group consisting of amylosucrase, glucan sucrose, cyclodextrin glycosyltranserase, and dextransucrase. It may be one or more enzymes selected from, but is not limited thereto.

In an exemplary embodiment, the isomaltose donor may be a saccharide containing at least one glucose molecule.

In an exemplary embodiment, the saccharide may be one or more saccharides selected from the group consisting of disaccharides, trisaccharides, and oligosaccharides, but is not limited thereto.

In an exemplary embodiment, the reaction may include reacting by adding an isomaltose donor to an aqueous solution containing EGCG at a concentration of 0.1 to 2.0% (w/v) based on the total volume of the reaction solution. Specifically, the concentration of the EGCG aqueous solution is 0.1% (w / v) or more, 0.2% (w / v) or more, 0.3% (w / v) or more, 0.4% (w / v) or more, 0.5% (w / v) or more v) greater than or equal to 0.6% (w/v), greater than or equal to 0.7% (w/v), greater than or equal to 0.8% (w/v), greater than or equal to 0.9% (w/v), or greater than or equal to 1.0% (w/v); , 1.1% (w / v) or less, 1.2% (w / v) or less, 1.3% (w / v) or less, 1.4% (w / v) or less, 1.5% (w / v) or less, 1.6% (w /v) or less, 1.7% (w/v) or less, 1.8% (w/v) or less, 1.9% (w/v) or less, or 2.0% (w/v) or less.

In an exemplary embodiment, the reaction may include reacting by adding an enzyme at a concentration of 0.01 to 10 units/mL based on the total volume of the reaction solution. Specifically, the concentration of the enzyme added to the method is 0.01 unit / mL or more, 0.02 unit / mL or more, 0.03 unit / mL or more, 0.04 unit / mL or more, 0.05 unit / mL or more, 0.06 unit / mL or more, 0.07 unit /mL or more, 0.08 unit/mL or more, 0.09 unit/mL or more, 0.1 unit/mL or more, 0.2 unit/mL or more, 0.3 unit/mL or more, 0.4 unit/mL or more, 0.5 unit/mL or more, 0.6 unit/mL More than 0.7 unit/mL, more than 0.8 unit/mL, more than 0.9 unit/mL, more than 1.0 unit/mL, more than 1.5 unit/mL, more than 2.0 unit/mL, more than 2.5 unit/mL, more than 3.0 unit/mL, 3.5 unit/mL or more, 4.0 unit/mL or more, 4.5 unit/mL or more, or 5.0 unit/mL or more, but 5.5 unit/mL or less, 6.0 unit/mL or less, 6.5 unit/mL or less, 7.0 unit/mL or less; 7.5 unit/mL or less, 8.0 unit/mL or less, 8.5 unit/mL or less, 9.0 unit/mL or less, 9.5 unit/mL or less, or 10 unit/mL or less.

In an exemplary embodiment, the reaction may include reacting in the presence of 1 to 50% (v/v) of an organic solvent based on the total volume of the reaction solution. That is, an organic solvent may be further included as a solvent other than water. Specifically, the organic solvent is 1% (v / v) or more, 10% (v / v) or more, 15% (v / v) or more, 20% (v / v) or more based on the total solution volume, or 25% (v/v) or more, but 30% (v/v) or less, 35% (v/v) or less, 40% (v/v) or less, 45% (v/v) or less, or 50% ( v/v) or less.

In an exemplary embodiment, the reaction may be to react the EGCG, the isomaltose donor, and the enzyme for 0.1 to 48 hours. Specifically, the reaction time is 0.1 hour or more, 1 hour or more, 5 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, or 25 hours or more, based on conversion of 90% or more of EGCG diglucoside from EGCG. In addition, EGCG, the isomaltose donor, and the enzyme may be reacted for 30 hours or less, 35 hours or less, 40 hours or less, 45 hours or less, or 48 hours or less.

In an exemplary embodiment, the reaction may be performed at a temperature of 25 to 50 °C. Specifically, the reaction temperature may be 25 °C or higher, 30 °C or higher, or 35 °C or higher, and 40 °C or lower, 45 °C or lower, or 50 °C or lower.

In an exemplary embodiment, the reaction may be performed under the control of pH 3 to 8 of the solution. Specifically, during the reaction, the pH of the solution may be adjusted to 3 or more, 4 or more, or 5 or more, and 6 or less, 7 or less, or 8 or less.

In an exemplary embodiment, the composition may be an aqueous solution composition, characterized in that the remaining life of the glycoside compound in the composition is 40 to 60 hours (h) when calculated by the following three-step formula.

(a) Step 1: Apply the reaction rate equation

Here, A is the measurement quality characteristic, K is the reaction rate constant, n is the reaction order, and t is the elapsed time.

(b) Step 2: Apply the Arrhenius Equation

아레니우스 방정식에 자연로그를 적용하여 다음 수식으로 전환한다.

Applying the natural logarithm to the Arrhenius equation converts to the following equation.

Here, A is the Arrhenius constant, E _a is the activation energy (cal/mol), R is the gas constant (1.987 cal/mol), T is the absolute temperature, and K is the reaction rate constant.

(c) Step 3: Calculate remaining life,t

By arranging the LnK obtained in step 2 and the first-order reaction equation in step 1, the following formula is obtained.

Specifically, the remaining life may be 40 hours or more, 45 hours or more, or 50 hours or more, and 55 hours or less, or 60 hours or less when measured in an aqueous solution at 25 ° C.

Hereinafter, the present invention will be described in more detail through examples and the like. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples. Anything that has substantially the same configuration and achieves the same effect as the technical concept described in the claims of the present invention is included in the technical scope of the present invention.

Example

(One) Preparation of EGCG diglucoside

As shown in FIG. 1, EGCG and sucrose were reacted with an enzyme in an aqueous solution to prepare EGCG diglucoside modified with isomaltose at the 4'-OH position of the EGCG molecule, and analyzed.

(2) Isolation and purification of EGCG diglucoside

To analyze EGCG diglucoside, the enzyme reactant was separated and purified into a single substance of high purity (MPLC and prep-HPLC), and it was confirmed that the substance was a single substance by a method for confirming the purity of the substance (HPLC-PDA, Waters Corp.) (FIG. 2). .

(3) Structural analysis of EGCG diglucoside

It was confirmed by mass spectrometry (FIG. 3) and high-resolution mass spectrometry (FIG. 4) that two glucose molecules were bound to EGCG in the prepared EGCG diglucoside. In addition, the structural characteristics of the molecule were confirmed by nuclear magnetic resonance analysis, and the intra-molecular and intra-glucose bonding forms (FIG. 5). The binding structure of the molecule is EGCG 4'-O-α-D-Glc (1"→6")-α-D-Glc (EGCG 4'-O-α-D-isomaltose).

(4) Aqueous stability evaluation of EGCG diglucoside

The stability evaluation of the aqueous phase was accelerated at three different equal interval temperatures to evaluate the remaining amount, and the remaining life at a specific temperature was predicted using the Arrhenius equation (Table 1). As a result, the stability of the aqueous phase was improved by about two times compared to the parent molecule, EGCG.

The decrease in residual amount with time at the three temperatures obtained by the accelerated life evaluation of EGCG and EGCG diglucoside was explained by the first order reaction, and showed a very good correlation with the amount of decrease with time. The linear correlation between the slope and temperature of the graphs obtained at the three temperatures was very good (FIG. 6).

Life prediction was performed in three stages as follows.

(a) Step 1: Apply the reaction rate equation

Here, A is the measurement quality characteristic, K is the reaction rate constant, n is the reaction order, and t is the elapsed time.

(b) Step 2: Apply the Arrhenius Equation

아레니우스 방정식에 자연로그를 적용하여 다음 수식으로 전환한다.

Applying the natural logarithm to the Arrhenius equation converts to the following equation.

Here, A is the Arrhenius constant, E _a is the activation energy (cal/mol), R is the gas constant (1.987 cal/mol), T is the absolute temperature, and K is the reaction rate constant.

(c) Step 3: Calculate remaining life,t

By arranging the LnK obtained in step 2 and the first-order reaction equation in step 1, the following formula is obtained.

The remaining life of EGCG diglucosde was improved by about 2 times compared to EGCG in aqueous solution at 25 ° C (Fig. 7).

Claims (15)

An EGCG glycoside compound in which 4'-OH of epigallocatechin gallate (EGCG) is substituted with isomaltose, a salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof. According to claim 1,
The glycoside is an EGCG diglucoside represented by Formula 1 below, an EGCG glycoside compound, a salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof.
[Formula 1]

An aqueous solution composition comprising the EGCG glycoside compound of claim 1 or 2, a salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof. According to claim 3,
The composition is a food, cosmetic, or pharmaceutical composition, an aqueous solution composition. A method for producing the EGCG glycoside compound according to claim 1, a salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof,
The method comprises reacting EGCG and an isomaltose donor in the presence of an enzyme;
The enzyme is a glycosyl hydrolase (glycosyl hydrolase) family of enzymes, manufacturing method. According to claim 5,
The glycosyl hydrolase enzyme is at least one enzyme selected from the group consisting of amylosucrase, glucan sucrose, cyclodextrin glycosyltranserase, and dextransucrase,
manufacturing method. According to claim 5,
The method of producing EGCG diglucoside, wherein the isomaltose donor is a saccharide containing at least one glucose molecule. According to claim 7.
The saccharide is at least one saccharide selected from the group consisting of disaccharides, trisaccharides, and oligosaccharides,
manufacturing method. According to claim 5,
The reaction comprises reacting by adding an isomaltose donor to an aqueous solution containing EGCG at a concentration of 0.1 to 2.0% (w / v) based on the total volume of the reaction solution.
manufacturing method. According to claim 5,
The reaction comprises reacting by adding the enzyme at a concentration of 0.01 to 10 units / mL based on the total volume of the reaction solution.
manufacturing method. According to claim 5,
The reaction comprises reacting under an organic solvent of 1 to 50% (v / v) based on the total volume of the total reaction solution,
manufacturing method. According to claim 5,
The reaction is to react the EGCG, isomaltose donor and enzyme for 0.1 to 48 hours,
manufacturing method. According to claim 5,
The reaction is made at a temperature of 25 to 50 ℃,
manufacturing method. According to claim 5,
The reaction is carried out under the control of pH 3 to 8 of the solution,
manufacturing method. According to claim 3,
The composition is characterized in that the remaining life of the glycoside compound in the composition is 40 to 60 hours (h) when calculated by the following three-step formula, an aqueous solution composition.
(a) Step 1: Application of the reaction rate equation

Here, A is the measurement quality characteristic, K is the reaction rate constant, n is the reaction order, and t is the elapsed time.
(b) Step 2: Application of the Arrhenius equation

Applying the natural logarithm to the Arrhenius equation converts to the following equation.

Here, A is the Arrhenius constant, E _a is the activation energy (cal/mol), R is the gas constant (1.987 cal/mol), T is the absolute temperature, and K is the reaction rate constant.
(c) Step 3: Calculate remaining life,t
By arranging the LnK obtained in step 2 and the first-order reaction equation in step 1, the following formula is obtained.

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